Radionuclide labeling of vitamin B12 and coenzymes thereof

ABSTRACT

A compound useful for in vivo imaging of organs and tumors is provided of formula: ##STR1## wherein ##STR2## is a cobalamin, ##STR3## is derived from a corrin carboxylic acid group of said cobalamin, Y is a linking group and X is a chelating group, optionally comprising a detectable radionuclide or a paramagnetic metal ion, and n is 1-3.

BACKGROUND OF THE INVENTION

For several years after the isolation of vitamin B₁₂ as cyanocobalaminin 1948, it was assumed that cyanocobalamin and possiblyhydroxocobalamin, its photolytic breakdown product, occurred in man.Since then it has been recognized that cyanocobalamin is an artifact ofthe isolation of vitamin B12 and that hydroxocobalamin and the twocoenzyme forms, methylcobalamin and adenosylcobalamin, are the naturallyoccurring forms of the vitamin.

The structure of these various forms is shown in FIG. 1, wherein X isCN, OH, CH₃ or adenosyl, respectively. Hereinafter, the term cobalaminwill be used to refer to all of the molecule except the X group. Thefundamental ring system without cobalt (Co) or side chains is calledcorrin and the octadehydrocorrin is called corrole. The Co-contgheptacarboxylic acid resulting from hydrolysis of all the amide groupswithout the CN and the nucleotide, is designated cobyrinic acid. Thecorresponding hexacarboxylic acid with D-1-amino-2-propanol side chain fis called cobinic acid and the hexacarboxylic acid with theα-D-ribofuranose-3-phosphate attached to the 2-position of the aminopropanol is called cobamic acid. Thus, cobamide is the hexaamide ofcobamic acid, cobyric acid is the hexaamide of cobyrinic acid andcobinamide is the hexaamide of cobinic acid. FIG. 1 is adapted from TheMerck Index, Merck & Co. (11th ed. 1989), wherein X is above the planedefined by the corrin ring and nucleotide is below the plane of thering. The corrin ring has attached six amidoalkyl (H₂ NC(O)Alk)substituents, at the 2, 3, 7, 8, 13, and 18 positions, which can bedesignated a-e and g, respectively. See D. L. Anton et al., J. Amer.Chem. Soc., 102, 2215 (1980). The molecule shown in FIG. 1 can beabbreviated as shown below: ##STR4## wherein, e.g., X is CN, OH, CH₃, oradenosyl.

Methylcobalamin serves as the cytoplasmic coenzyme for ⁵N-methyltetrahydrofolate:homocysteine methyl transferase (methioninesynthetase, EC 2.1.1.13), which catalyzes the formation of methioninefrom homocysteine. Adenosylcobalamin is the mitochondrial coenzyme formethylmalonyl CoA mutase (EC5.4.99.2) which interconverts methylmalonylCoA and succinyl CoA.

All forms of vitamin B₁₂ (adenosyl-, cyano-, hydroxo-, ormethylcobalamin) must be bound by the transport promins, IntrinsicFactor and Transcobalamin II to be biologically active. Specifically,gastrointestinal absorption of vitamin B₁₂ relies upon the intrinsicfactor-vitamin B₁₂ complex being bound by the intrinsic factor receptorsin the terminal ileum. Likewise, intravascular transport and subsequentcellular uptake of vitamin B₁₂ throughout the body is dependent upontranscobalamin II and the cell membrane transcobalamin II receptors,respectively. After the transcobalamin II-vitamin B₁₂ complex has beeninternalized, the transport protein undergoes lysozymal degradation,which releases vitamin B₁₂ into the cytoplasm. All forms of vitamin B₁₂can then be interconverted into adenosyl-, hydroxo-, or methylcobalamindepending upon cellular demand. See, for example, A. E. Finkler et al.,Arch. Biochem. Biophys., 120, 79 (1967); C. Hall et al., J. CellPhysiol., 133, 187 (1987); M. E. Rappazzo et al., J. Clin. Invest., 51,1915 (1972) and R. Soda et al., Blood, 65, 795 (1985).

Cells undergoing rapid proliferation have been shown to have increaseduptake of thymidine and methionine. (See, for example, M. E. vanEijkeren et al., Acta Oncologica, 31, 539 (1992); K. Kobota et al., J.Nucl. Med., 32, 2118 (1991) and K. Higashi et al., J. Nucl. Med., 34,773 (1993)). Since methylcobalamin is directly involved with methioninesynthesis and indirectly involved in the synthesis of thymidylate andDNA, it is not surprising that methylcobalamin as well asCobalt-57-cyanocobalamin have also been shown to have increased uptakein rapidly dividing tissue (for example, see, B. A. Cooper et al.,Nature, 191, 393 (1961); H. Flodh, Acta Radiol. Suppl., 284, 55 (1968);L. Bloomquist et al., Experientia, 25, 294 (1969)). Additionally,upregulation in the number of transcobalamin II receptors has beendemonstrated in several malignant cell lines during their acceleratedthymidine incorporation and DNA synthesis (see, J. Linderoans et al.,Exp. Cell. Res., 184, 449 (1989); T. Amagasaki et al., Blood, 26, 138(1990) and J. A. Begly et al., J. Cell Physiol., 156, 43 (1993).

Vitamin B₁₂ has several characteristics which potentially make it anattractive in vivo tumor imaging agent. Vitamin B₁₂ is water soluble,has no known toxicity, and in excess is excreted by glomerularfiltration. In addition, the uptake of vitamin B₁₂ could potentially bemanipulated by the administration of nitrous oxide and otherpharmacological agents (D. Swanson et al., Pharmaceuticals in MedicalImaging, MacMillan Pub. Co., N.Y. (1990) at pages 621-628).

Bacteria naturally insert Cobalt-59 into the corrin ring of vitamin B₁₂.Commercially this has been exploited by the fermentative production ofCo-56, Co-57, Co-58, and Co-60 radiolabeled vitamin B₁₂. For example,see Chaiet et al., Science, 111, 601 (1950). Unfortunately Cobalt-57,with a half life of 270.9 days, makes Co-57-cyanocobalamin unsuitablefor clinical tumor imaging. Other metal ions (cobalt, copper and zinc)have been chemically inserted into naturally occurringdescobaltocorrinoids produced by Chromatium and Streptomyces olivaceous.Attempts to chemically insert other metal ions in these cobalt freecorrinoid rings has been unsuccessful. The placement of metals (cobalt,nickel, palladium, platinum, rhodium, zinc, and lithium) into asynthetic corrin ring has not presented any major difficulties. However,their instability and cost to produce makes them impractical forbiological assays. Although Co-59 is a weakly paramagnetic quadrapolarnuclei in the 2⁺ oxidation state, Co-59 exists in the 3⁺ oxidation statewithin the corrin ring of vitamin B₁₂ and is diamagnetic. Therefore,insertion of either a radioactive or paramagnetic metal ion other thancobalt into the corrin ring does not seem feasible at this time.

A process for preparing ¹²⁵ I-vitamin B₁₂ derivatives is described inNiswender et al. (U.S. Pat. No. 3,981,863). In this process, vitamin B₁₂is first subjected to mild hydrolysis to form a mixture ofmonocarboxylic acids, which Houts, infra, disclosed to contain mostlythe (e)-isomer. The mixture is then reacted with a p-(aminoalkyl)phenolto introduce a phenol group into the B₁₂ acids (via reaction with one ofthe free carboxylic acid groups). The mixed substituent B₁₂ derivativesare then iodinated in the phenol-group substituent. This U.S. patentteaches that the mixed ¹²⁵ I-B₁₂ derivatives so made are useful in theradioimmunoassay of B₁₂, using antibodies raised against the mixture.

T. M. Houts (U.S. Pat. No. 4,465,775) reported that the components ofthe radiolabelled mixture of Niswender et al. did not bind with equalaffinity to IF. Hours disclosed that radioiodinated derivatives of thepure monocarboxylic (d)-isomer are useful in assays of B₁₂ in which IFis used. However, although Hours generally discloses that themonocarboxylic (d)-isomer can be labelled with fluorophores or enzymesand used in competitive assays for vitamin B₁₂ in fluids, a continuingneed exists for labelled vitamin B₁₂ derivatives suitable for tumor andorgan imaging and therapy.

SUMMARY OF THE INVENTION

The present invention provides detectable compounds of the generalformula (I): ##STR5## wherein the moiety ##STR6## is cobalamin, X is CN,OH, methyl or adenosyl, ##STR7## is the residue of a monocarboxylic acidof the cobalamin, derived from a corrin propionamide group, and ispreferably the essentially pure (b)-, (d)-, or (e)- monocarboxylic acid;Y is a linking group and Det is a chelating group comprising adetectable metal, such as a radionuclide or paramagnetic metal ion.Preferably, the linking group is --N(H)(CH₂)₂₋₆ NH--.

For example, compounds of formula (I) derived from the(b)-monocarboxylic acid, wherein Det is thediethylenetriaminepentaacetic acid group (DTPA), were preparedcomprising Tc-99n, In-111 and Gd-153. These compounds were found to bereadily absorbed through the mammalian peritoneal membrane andgastrointestinal tract, to localize within the liver, kidney, pancreas,and spleen. Therefore, the present compounds can be used to evaluatehepatic, splenic, renal, pancreatic, and small bowel function in mammalssuch as humans and experimental animals, by administering a compound offormula (I) to the mammal and detecting its presence in the targetorgan, using appropriate normal control values for comparison.

Certain neoplastic tissue has been found to act as a vitamin B₁₂ sink,accumulating the vitamin to a greater extent than the surrounding slowerdividing tissue. Therefore, the present compounds can also be used fortumor imaging and/or targeted cancer therapy, by administering acompound of formula (I) to a mammal afflicted with a tumor, so that thecompound localizes in the tumor, and optionally, detecting the presenceof the compound in the tumor, particularly tumors of the organs listedabove.

Intermediates useful in the preparation of the compounds of formula (I)are also an aspect of the invention, including compounds wherein Det isreplaced by Chel, which is an organic chelating group, or chelator,capable of chelating a radionuclide or radioisotope.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the structure of vitamin B₁₂, wherein X is CN (cyano),OH, CH₃ or adenosyl.

FIG. 2 schematically depicts the synthesis of a cobalamin metal ion DTPAcomplex.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of formula I can be prepared by producing a monocarboxylicacid of X- cobalamin!, wherein X is cyano-, methyl, adenosyl, and thelike. These compounds can be prepared by the mild acid hydrolysis ofcyanocobalamin, which has been shown to yield a mixture of mono-, adicarboxylic acids and one tricarboxylic acid. These carboxylic acidsare derived from the propionamide side chains designated b, d and e, asdiscussed hereinabove, which are more susceptible to hydrolysis than theamide groups on acetamide side chains a, c, and g. The (b)-, (d)-, and(e)-monocarboxylic acids can be separated by column chromatography. SeeFIG. 1 herein, and FIG. 1 of D. L. Anton et al., J. Amer. Chem. Soc.,102, 2215 (1980). See, also, J. B. Armitage et al., J. Chem. Sot., 3349(1953); K. Bernhauer, Biochem. Z., 344, 289 (1966); H. P. C. Hogenkampet al., Biochemistry, 14, 3707 (1975); and L. Ellenbogen, in"Cobalamin," Biochem. and Pathophysiol., B. Babior, ed., Wiley, N.Y.(1975) at chapter 5.

The X- cobalamin! CO₂ H! can be linked to the metal chelator by means ofa linking group, which is preferably a divalent, or "bifunctional"organic linking group. Such linking groups comprise two reactive groups,one that is coupled to the CO₂ H group, and the other that is coupled tothe metal chelator. A variety of homobifunctional and heterobifunctionallinking reagents known in the art are useful in the present invention.Preferred linkers comprise one or two amino or hydroxyl groups, such asω-aminoalkanoic acids, e.g., ε-amino caproic acid (H₂ N--(CH₂)₅ --COOH),or alkane diamines including 1,4-diaminobutane, 1,5-diaminopentane and1,6-diaminohexane, and the like. Particularly preferred among theaminoalkanoic acids and similar compounds are those which are soluble inaqueous buffers.

Det is a chelating group comprising a radionuclide, such as a metallicradioisotope. Preferred among these chelating compounds "chelators" or(chel) are such polycarboxylic acids as EDTA, DTPA, DCTA, DOTA, TETA, oranalogs or homologs thereof.

DTPA (diethylenetriaminepentaacetic acid) can be attached to cobalamincarboxylic acid(s) via reaction of diethylenetriaminepentaaceticdianhydride (Aldrich Chem. Co.) with a linker comprising a free aminogroup. This yields a Chel group that is2-(amidomethyl)-1,1,7,7-diethylenetriaminetetraacetic acid. Thischelator can be reacted with radionuclides to yield a Det moiety of thegeneral formula ##STR8## wherein M is the radionuclide. The syntheticroute to a cobalamin metal ion DTPA complex (4) is schematically shownin FIG. 2, wherein WSC=water soluble carbodiimide.

The chelator (chel) DCTA has the general formula: ##STR9##

DCTA is a cyclohexane-based metal chelator, wherein R³ may by (C₁-C₄)alkyl or CH₂ CO₂ --, which may be attached to the Y throughpositions 4 or 5, or through the group R³ and which carries from 1 to 4detectable metal or nonmetal cations (M), monovalent cations, or thealkaline earth metals. Thus, with metals of oxidation state +1, eachindividual cyclohexane-based molecule may carry up to 4 metal cations(where both R³ groups are CH₂ COOM). As is more likely, with higheroxidation states, the number of metals will decrease to 2 or even 1 percyclohexane skeleton. This formula is not intended to limit the moleculeto any specific stereochemistry. In particular, both areincfunctionalities may be either cis or trans to each other.

Other macrocyclic carboxylic acid chelators which can be linked to thecobalamin carboxylic acid via bis-amino linking groups include TETA1,4,8,11-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid;1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA);1,4,8,12-tetraazacyclopentadecane-N,N',N",N'"-tetraacetic acid (15N4);1,4,7-triazacyclononane-N,N',N"-triacetic acid (9N3); and1,5,9-triazacyclododecane-N,N',N"-triacetic acid (12N3). Bifunctionalchelators based on macrocyclic ligands in which conjugation is via anactivated arm attached to the carbon backbone of the ligand can beemployed as described by M. Moi et al., J. Amer. Chem., Soc., 49, 2639(1989)(2-p-nitrobenzyl-1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraaceticacid); S. V. Deshpande et al., J. Nucl. Med., 31, 473 (1990); G. Kuseret al., Bioconj. Chem., 1, 345 (1990); C. J. Broan et al., J. C. S.Chem. Comm., 23, 1739 (1990); and C. J. Anderson et al., J. Nucl. Med.36, 850 (1995)(6-bromoacetamido-benzyl-1,4,8,11-tetraazacyclotetadecane-N,N',N",N'"-tetraaceticacid (BAT)).

Any metal capable of being detected in a diagnostic procedure in vivo orin vitro can be employed as M in the Det moieties. Particularly, anyradioactive metal ion capable of producing a diagnostic result in ahuman or animal body or in an in vitro diagnostic assay may be used inthe practice of the present invention. Suitable ions include thefollowing: Antimony-124, Antimony-125, Arsenic-74, Barium-103,Barium-140, Beryllium-7, Bismuth-206, Bismuth-207, Cadmium- 109,Cadmium-115m, Calcium-45, Cerium- 139, Cerium-141, Cerium-144,Cesium-137, Chromium-51, Cobalt-56, Cobalt-57, Cobalt-58, Cobalt-60,Cobalt-64, Erbium-169, Europium-152, Gadolinium-153, Gold-195, Gold-199,Hafnium-175, Hafnium-175-181, Indium-111, Iridium-192, Iron55, Iron-59,Krypton-85, Lead-210, Manganese-54, Mercury-197, Mercury-203,Molybdenum-99, Neodymium-147, Neptunium-237, Nickel-63, Niobiumo-95,Osmium-185+191, Palladium-103, Platinum-195m, Praseodymium-143,Promethium-147, Protactinium-233, Radium-226, Rhenium-186, Rubidium-86,Ruthenium103, Ruthenium-106, Scandium-44, Scandium-46, Selenium-75,Silver-110m, Silver-111, Sodium-22, Strontium-85, Strontium-89,Strontium-90, Sulfur-35, Tantalum-182, Technetium-99m, Tellurium- 125,Tellurium-132, Thallium-204, Thorium-228, Thorium-232, Thallium- 170,Tin-113, Titanium-44, Tungsten-185, Vanadium-48, Vanadium-49,Ytterbium-169, Yttrium-88, Yttrium-90, Yttrium-91, Zinc-65, andZirconium-95.

The compounds of formula (I) are preferable dissolved or dispersed in anontoxic liquid vehicle, such as physiological saline or a similaraqueous vehicle, to the desired concentration. A preselected analytical,diagnostic or therapeutic unit dose is then administered to the testanimal or human patient, by oral administration or ingestion or byparenteral administration, as by intravenous or intraperitoneal infusionor injection, to attain the desired in vivo concentration. Doses usefulfor imaging or treating human organs or tumors can be derived, fromthose found to be effective to image or treat organs in humans in vitroor in animal models, such as those described hereinbelow, or fromdosages of other labelled vitamin B₁₂ molecules, previously employed inanimal therapy or imaging.

The invention will be further described by reference to the followingdetailed examples, wherein cyanocobalamin and1-ethyl-3-(3-dimethylaminopropyl) carbodiimide were purchased from SigmaChem. Co., St. Louis, Mo. Adenosine, 1,4-diaminobutane dihydrochloride,diethylenetriamine pentaacetic (DPTA), hexamethylphosphoramide,1-hydroxybenzotriazole hydrate, iodomethane and thionylchloride wereobtained from Aldrich Chem. Co., Milwaukee, Wis. Thin layerchromatography (TLC) silica gel and PET-cellulose sheets were purchasedfrom E. M. Science, Gibbstown, N.J. Tc^(99m) and In¹¹¹ were obtainedfrom Mallinckrodt Medical, Inc. and Gd¹⁵³ was obtained from Amersham.Other inorganic salts and solvents were obtained in the highest purityavailable.

UV-visible spectra were recorded on a Hewlett-Packard diode arrayspectrophotometer. DTPA dianhydride and 5'-chloro-5'-deoxyadenosine weresynthesized as described by W. C. Eckelman et al., J. Pharm. Sci., 64,704 (1975) and K. Kikugawa et al., Tetrahedron Lett., 87 (1971),respectively. The monocarboxylic acids of cyaaocobalamin,methylcobalamin-b-carboxylic acid and adenosylcobalamin-b-carboxylicacid were prepared and isolated as described by H. P. C. Hogenkamp,Biochemistry, 13, 2736 (1974); D. L. Anton et al., J. Amer. Chem. Soc.,102, 2215 (1980); R. H. Yamada et al., J. Biol. Chem., 247, 6266 (1972)and D. Dolphin, Methods in Enzymology, XVille, 34-52 (1971).Methylcobalamin, adenosylcobalamin and their derivatives are lightsensitive, especially in solution, and all reactions and manipulationswere carried out in the dark or in dim light.

All images for the in vivo studies were obtained on a GE 500 maxicamerausing a LEAP collimator with a 20% window about the 140 keV energy peakof technetium, and a medium energy collimator with a 20% window aboutthe 174 keV and 247 keV energy peaks of Indium. A 256×256 matrix with adedicated pinnacle computer system was used to collect and analyze thedata.

EXAMPLE 1 Cyanocobalamin-b-(4-aminobutyl)amide

A mixture containing cyanocobalamin-b-carboxylic acid (1.0 g, 0.6 mmol),hydroxybenzotriazole (0.81 g, 6 mmol) and 1,4-diaminobutanedihydrochloride (4.8 g, 30 mmol) in 100 ml of water was adjusted to pH7.8. 1-Ethyl-3-(3'-dimethylaminopropyl)carbodiimide (1.26 g, 6.6 mmol)was then added, the pH was adjusted to 6.4 and the reaction stirred atroom temperature for 24 h. TLC on silica gel using n-butanol-acetic acidwater (5:2:3) showed the reaction to be complete.Cyanocobalamin-b-(4-aminobutyl)amide was extracted into 92% aqueousphenol and the phenol layer was washed several times with equal volumesof water. To the phenol extract were added 3 volumes of diethylether and1 volume of acetone. The desired cobalamin was removed from the organicphase by several extractions with water. The combined aqueous layerswere extracted three times with diethylether to remove residual phenol,concentrated to approximately 20 ml in vacuo and crystallized fromaqueous acetone. Yield 955 mg, 92%.

EXAMPLE 2 Cyanocobalamin-b-(4-aminobutyl)amide DTPA

Cyanocobalamin-b-(4-aminobutyl) amide (500 mg), 0.3 mmol) was dissolvedin 30 ml sat. sodium bicarbonate and treated with solid DTPA dianhydride(1.2 g, 3.4 mmol). The progress of the reaction was monitored by TLC onPEI plates using n-butanol-acetic acid-water (5:2:3) as the solvent.After 30 min incubation at room temperature a second 1.2 g of thedianhydride was added. After two additional additions of dianhydridewith adjustments of the pH to 8.2 the reaction mixture was incubatedovernight. Cyanocobalamin-DPTA adduct was then extracted into 92%aqueous phenol and purified as described above. The preparation wasevaporated to dryness in vacuo and isolated as a glass. Yield 460 mg,77%. The cyanobalamin-DTPA adduct behaves as a polyanion on paperelectrophoresis in 0.1M sodium phosphate buffer pH 7.1.

EXAMPLE 3 Methylcobalamin-b-(4-aminobutyl)amide

Methylcobalamin-b-carboxylic acid (1.0 g, 0.6 mmol) was reacted withdiaminobutane dihydrochloride as described above for the cyanoderivative. The cobalamin was purified by extraction through phenol (seeabove) and the resulting aqueous solution was concentrated in vacuo.This solution was chromatographed on AG1-X2 200-400 mesh in the acetateform (20×2.5 cm) and the pass through collected. The pass through wasconcentrated to approximately 20 ml and the desired cobalamincrystallized from aqueous acetone. Yield 920 mg, 88%. Unreactedmethylcobalamin-b-carboxyclic acid was eluted with 1M acetic acid,concentrated and crystallized from aqueous acetone. Yield 60 mg, 6%.

EXAMPLE 4 Methylcobalamin-b-(4-aminobutyl)amide DTPA

Methylcobalamin-b-(4-aminobutyl)amide (500 mg, 0.3 mmol) was dissolvedin 30 ml saturated sodium bicarbonate and reacted with solid DTPAdianhydride as described above. The methyl cobalamin-DTPA adduct waspurified by extraction through phenol, evaporated to dryness in vacuoand isolated as a glass. Yield 600 mg, 96%.

EXAMPLE 5 Adenosylcobalamin-b-(4-aminobutyl)amide

Adenosylcobalamin-b-carboxylic acid (500 mg, 0.3 mmol) was reacted withdiaminobutane dihydrochloride (2.4 mg, 15 mmol) as described above. Thecobalamin was purified by extraction through phenol (see above). Theresulting aqueous solution was concentrated in vacuo and applied toAG-50 X2, 200-400 mesh, in the hydrogen form (20×25 cm). The column waswashed thoroughly with water to remove hydroxybenzotriazole and thedesired cobalamin eluted with 1M ammonium hydroxide. After an additionalextraction through phenol, adenosylcobalamin-b-(4-aminobutyl)amide wasisolated as a glass. Yield 366 mg, 77%.

EXAMPLE 6 Adenosylcobalamin-b-(4-aminobutyl)amide DTPA

Adenosylcobalamin-b-(4-aminobutyl)amide (366 mg, 0.23 mmol) wasdissolved in 30 ml saturated sodium bicarbonate and treated with solidDTPA dianhydride (1.0 g, 2.8 mmol) as described above. The cobalamin waspurified through phenol (see above). The resulting aqueous solution wasconcentrated and applied to AG-50 X2, 200-400 mesh, in the hydrogen form(6.0×2.5 cm), the column was washed with water and the desired cobalamineluted with 0.1M ammonium hydroxide. The solution was evaporated todryness in vacuo and adenosylcobalamin-b-(4-aminobutyl)amide DTPAisolated as a glass. Yield 400 mg, 80%.

EXAMPLE 7 Interaction with Intrinsic Factor and Transcobalamin Proteins

Under dim light, 1000 μg of the non-labeled methyl-. adenosyl-, andcyanocobalamin-b-(4-aminobutyl)amide-DTPA, as well as 1000 μg ofcyanocobalamin and DTPA (Sigma, St. Louis, Mo. 63178), were separatelydissolved in 10 ml of normal saline at room temperature. Each of thefive 1000 μg/10 ml samples were stored in sealed, aluminum-wrapped 10 mlvials to prevent exposure to light. No buffers were added to thesolutions. The pH of the solutions, measured by a Beckman 40 pH meter(Beckman Instruments, Fullerton, Calif.): Cyanocobalamin=5.75,DTPA=3.78; cyano, methyl and adenosylcobalamin-DTPA analogues were 5.75,6.10, and 6.19, respectively.

To assess in vitro binding to Intrinsic Factor (IF) and Transcobalamins(TC), the intrinsic factor blocking antibody (IFBA) and Unsaturatedvitamin B₁₂ Binding Capacity (UBBC) assays were performed with serumrandomly obtained from five patients being evaluated for perniciousanemia at the Mayo Clinic. The IFBA and UBBC assays were first performedfor clinical purposes as previously described by V. F. Fairbanks et al.,Mayo Clin. Proc., 58, 203 (1983); Intrinsic Factor Blocking Antibody ⁵⁷Co! Radioassay-Package insert, Diagnostic Products Corp.; D. Grossowiczet al., Proc. Exp. Biol., 109, 604 (1962) and C. Gottlieb et al., Blood,25, 6 (1965).

Next, the serum from the same five patients underwent modified IFBA andUBBC assays. Specifically, 1 μl of the five previously describedsolutions were separately incubated with purified IF or serum, topotentially saturate all IF and TC binding sites. After incubation for20 minutes at room temperature and for another 20 minutes at 4° C., 500μl of the stock (1000 μg/l) Cobalt-57-cyanocobalamin (MallinckrodtMedical, Inc., St. Louis, Mo. 63134) solution was added and the usualIFBA and UBBC protocols were then followed. All supernatant activity wascounted for four minutes on a gamma counter (Micromedix 10/20,Huntsville, Ala. 35805). The results are shown in Table I.

                                      TABLE I                                     __________________________________________________________________________       Clinical                                                                      Run  CNB.sub.12                                                                         MEB.sub.12 DTPA                                                                      ADB.sub.12 DTPA                                                                      CNB.sub.12 DTPA                                                                      DTPA                                        __________________________________________________________________________    UBBC                                                                          PT 1                                                                             741  <NSB 17.1   54.6   222.6  731.5                                       PT 2                                                                             632  <NSB 26.8   62.6   216.9  913.1                                       PT 3                                                                             2097 <NSB 278.9  590.3  713.3  2078.9                                      PT 4                                                                             1378 <NSB 60.9   126.9  433.2  1633.7                                      PT 5                                                                             1682 <NSB 91.1   163.9  643.2  1418.0                                      IFBA                                                                          PT 1                                                                             11942.5                                                                            951.5                                                                              4279   6758.5 5151   11899                                          (0.99)                                                                             (12.48)                                                                            (2.77) (2.30) (2.30) (0.99)                                      PT 2                                                                             11656                                                                              920.5                                                                              4082   6841.5 5133.5 11696.5                                        (1.02)                                                                             (12.90)                                                                            (2.92) (1.74) (2.31) (1.02)                                      PT 3                                                                             11780                                                                              912.5                                                                              4456.5 6828.5 5338.5 11735.5                                        (1.01)                                                                             (13.01)                                                                            (2.66) (1.74) (2.22) (1.01)                                      PT 4                                                                             11617                                                                              749  4414   7046.5 6002.5 11909                                          (1.02)                                                                             (15.85)                                                                            (2.69) (1.64) (1.98) (1.00)                                      PT 5                                                                             11653.5                                                                            858.5                                                                              4381.5 7096.5 5973.5 1178.5                                         (1.02)                                                                             (10.91)                                                                            (2.77) (1.72) (1.99) (1.02)                                      __________________________________________________________________________     NSB = Nonspecific binding; counts < 100 consistent with saturation of         transcobalamin proteins                                                       Negative reference for IFBA; no binding to intrinsic factor (<1.11)           Positive reference for IFBA; binding to intrinsic factor (>1.43)              Indeterminate reference value (1.11 → 1.43)                            Clinical Run = patients supernatant counts from UBBC and IFBA assays          DTPA = diethylenetriamine pentaacetic acid                                    CNB.sub.12 = cyanocobalamin                                                   MEB.sub.12 DTPA = methylcobalaminb-(4-aminobutyl)-amide-DTPA                  ADB.sub.12 DTPA = adenosylcobalaminb-(4-aminobutyl)-amide-DTPA                CNB.sub.12 DTPA = cyanocobalaminb-(4-aminobutyl)-amide-DTPA              

The IFBA assay demonstrated that DTPA does not significantly bind to IF(values less than the negative reference), whereas cyanocobalamin andthe cobalamin-DTPA analogs do, in varying degrees, competitively inhibitCo-57 cyanocobalamin from binding to intrinsic factor. By using thecounts of the Clinical run divided into the counts of the fivesolutions, the efficacy of binding to intrinsic factor can be estimated.The averaged percent binding of the five solutions to IF was:cyanocobalamin=92.5%; methylcobalamin-b-(4-aminobutyl)-amide-DTPA=63.2%;cyanocobalamin-b-(4-aminobutyl)-amide-DTPA=52.9%;adenosylcobalamin-b-(4-aminobutyl)-amide-DTPA =41.0% and 0.8% for DTPA.This is in contrast to the disclosure in Houts (U.S. Pat. No. 4,465,775)that the (b)-monocarboxylic acid of vitamin B₁₂ and its radioiodinatedderivative exhibit very low binding to IF.

Likewise the averaged percent binding of the five solutions to thetranscobalamin proteins was: cyanocobalamin=100%,methylcobalamin-b-(4aminobutyl)amide-DTPA=94.0%,adenosylcobalamin-b-(4-aminobutyl)amide-DTPA=DTPA =90.4%,cyanocobalamin-b-(4-aminobutyl)amide-DTPA =66.4% and 3.6% for DTPA.

Thus, the attachment of DTPA to vitamin B₁₂ does alter its binding tothe carrier proteins. As expected, non-labeled cyanocobalamin had thegreatest affinity for IF and the transcobalamin proteins.Methylcobalamin-b-(4-aminobutyl)amide-DTPA was next, followed byadenosylcobalamin-b-(4-aminobutyl)amide-DTPA, and finallycyanocobalamin-b-(4-aminobutyl)amide-DTPA. There was also somenonspecific binding of DTPA to the carrier proteins (0.8% and 3.6%).

EXAMPLE 8 Chelation of Radionuclides

Under dim light, 1000 μg of methyl-, adenosyl-, andcyanocobalamin-b-(4-aminobutyl)amide-DTPA were separately dissolved in200 μl of normal saline. Next, 500 μCi of Indium-111 or 250 μCi ofGadolinium-153 were added to the cobalamin-DTPA solutions. The reactionswere carried out at room temperature and room air. For the chelation oftechnetium, the dissolved cobalamin DTPA complexes were separatelyplaced into sealed 2 ml vials. Next, 200 μl of stannous chloridesolution (1000 μg/ml normal saline) were added to each vial. The vialswere purged with nitrogen gas for 5 minutes. After this time, 1-5 mCi ofTechnetium-99m was added to the N₂ purged vials. Each vial underwentfurther nitrogen purging for 5 minutes. All chelation reactions weremixed gently for 5 minutes.

Control mixtures of 1000 μg of cyanocobalamin were dissolved in 200 μlof normal saline. Cyanocobalamin was mixed with Tc-99m at roomtemperature and room air, as well as within nitrogen purged vialscontaining 200 μl of the described stannous chloride solution.Additionally, the cobalamin-DTPA complexes underwent Tc-99m labeling inopen vials at room air in the absence of the stannous chloride.

Specific activity was assessed by mixing 100 μl aliquots of methyl andadenosyl cobalamin-b-(4-aminobutyl)amide-DTPA (5 μg/100 μl normalsaline) with 50 μl stannous chloride solution (1 μg/50 μl normal saline)in nitrogen purged 2 ml vials. Technetium-99m in 10, 25, 50, 75, and 100mCi allotments of activity were added to the vials. The vials underwentgentle mixing and continuous nitrogen purging for five minutes after theaddition of technetium.

Efficiency of chelation and specific activity were assessed via thinlayer chromatography (TLC). Thin layer chromatographic strips (Grade 31ET Chr-thickness 0.50 mm, flow rate (water) 225 mm/30 min, Whatman LabSales, Hilsboro, Oreg. 97123) were developed in acetone in dim light.The dry strips were placed on film (Ektascan-MC1, Eastern Kodak,Rochester, N.Y. 14650) for autoradiography (AR). Chromatographic andautoradiographic results were visually compared. All the radiolabeledcobalamin-DTPA complexes underwent TLC and AR to confirm 100% labelingprior to in vivo administration.

Under acetone development, free Tc-99m migrates to the top of thechromatographic strip, whereas In-111 and Gd-153 diffusely spread overthe lower two-thirds of the strip. TLC and AR analysis demonstrated thatthere was 100% labeling of all three cobalamin-DTPA complexes withTc-99m, In-111, and Gd-153. Specifically, all radioactivity was confinedto the chromatographic distribution of the cobalamin analogues.

Since methyl and adenosyl cobalamin could potentially have greateruptake in malignant tissue, the chelation of Tc-99m, In-111, and Gd-153by methyl and adenosylcobalamin-b-(4-aminobutyl)amide-DTPA underwentgreater scrutiny. The chromatographic and autoradiographic images wereconsistently coincident. In contrast, modified cyanocobalamin did notdemonstrate any affinity for binding the three radionuclides. Asexpected, there was minimal labeling of the cobalamin-DTPA complexeswith Tc-99m in the absence of stannous chloride and hypoxic conditions.

At a concentration of 5 μg/100 μl the red color of the cobalamin-DTPAanalogues is barely discernible in the aqueous state, and undetectableon TLC. However, the AR distribution is the same when compared to themore concentrated cobalamin analogue solutions with lower specificactivity. Methyl and adenosyl cobalamin-b-(4-aminobutyl)amide-DTPA canchelate up to 50 mCi of technetium-99m per 5 μg with 100% efficiency.This results in a specific activity of 10 mCi/μg for the cobalamin-DTPAanalogue.

EXAMPLE 9 In Vivo Studies

A. Biodistribution: Methylcobalamin-b-(4-aminobutyl)amide-DTPA in aconcentration of 300 μg/100 μl normal saline was labeled with 3 mCi ofIndium-111. The labeled vitamin B₁₂ analogue was diluted with normalsaline to a final volume of 1000 μl. Via intraperitoneal injection (IP),five 12 week old female Balb-C mice (Harlan, Sprague, Dawley,Indianapolis, Ind. 46229) each received 200 μl (500 μCi) of themethylcobalamin-DTPA-¹¹¹ In complex. For comparison, Indium-111-DTPAhaving the same concentration and specific activity of themethylcobalamin-DTPA analogue, was injected IP into three mice. All micewere sacrificed at 24 hours via CO₂ inhalation. The pancreas, spleen,kidneys, and heart were dissected in their entirety. A portion of theliver, lung, left quadricep muscle, and flank fat were also harvested.All tissue samples and organs were weighed wet, minced in 2.0 ml normalsaline, and counted for five minutes in a gamma well counter (MinaxiAutogamma 5000, Packard Instrument, Downers Grove, Ill. 60515).

B. Gastrointestinal Absorption: Methylcobalamin-b-(4-aminobutyl)-DTPAand DTPA alone were labeled as described above, with the exception thatthe 3 mCi Indium/300 μg/100 μl normal saline solutions were not diluted.Two groups of three mice had a few drops of either ¹¹¹ In-DTPA ormethylcobalamin-b-(4-aminobutyl)-DTPA-In-111 placed in their oralcavities. The mice were sacrificed at 24 hrs, dissected, and studied asdescribed above.

A modified Schillings test was performed on two mice. Specifically, eachmouse received via subcutaneous and intraperitoneal administration, a1000 μg loading dose of non-labeledmethylcobalamin-b-(4-aminobutyl)amide-DTPA analogue. At 24 hrs, the micewere fed 2-3 drops of Indium-labeledmethylcobalamin-b-(4-aminobutyl)amide-DTPA-complex. Urine and feces werecollected from the three groups of mice after oral administration. Themice were sacrificed at 24 hours after ingestion of tracer and imagesand biodistribution data were obtained at that time.

C. Tumor Imaging: At 24 hours, there was a significant amount ofadenosylcobalamin-b-(4-aminobutyl) amide-DTPA-In-111 uptake within thetransplanted sarcoma both visually and by gamma well counting (TableII).

                                      TABLE II                                    __________________________________________________________________________         Kidney                                                                             Liver                                                                              Spleen                                                                             Pancreas                                                                           Heart                                                                              Lung Fat  Muscle                                                                             Tumor                            __________________________________________________________________________    Mouse 1                                                                            3717.5                                                                             943.3                                                                              433.1                                                                              304.2                                                                              134.7                                                                              130.9                                                                              101.4                                                                              93.6 --                               Mouse 2                                                                            3299.5                                                                             823.4                                                                              405.3                                                                              319.9                                                                              189.4                                                                              180.1                                                                              147.3                                                                              51.4 --                               Mouse 3                                                                            3462.7                                                                             768.6                                                                              366.8                                                                              310.3                                                                              171.2                                                                              113.1                                                                              102.8                                                                              43.9 --                               Mouse 4                                                                            224.0                                                                              56.9 44.1 13.4 10.3 6.2  12.6 5.4  --                               Mouse 5                                                                            130.2                                                                              41.5 26.2 13.0 3.9  6.0  19.5 5.6  --                               Mouse 6                                                                            281.6                                                                              66.1 57.7 14.1 12.5 10.5 18.8 5.0  --                               Mouse 7                                                                            621.4                                                                              126.4                                                                              67.8 40.0 35.0 38.4 --   13.6 --                               Mouse 8                                                                            700.5                                                                              111.7                                                                              66.6 29.3 29.8 51.2 --   12.4 --                               Mouse 9                                                                            601.7                                                                              115.8                                                                              66.3 41.2 31.3 40.6 --   12.0 --                               Mouse 10                                                                           119.4                                                                              24.0 19.5 6.0  5.6  5.4  --   8.9  --                               Mouse 11                                                                           117.3                                                                              25.5 19.0 6.7  5.0  5.3  --   2.6  --                               Mouse 12                                                                           110.1                                                                              23.2 18.1 5.9  4.8  5.0  --   3.7  --                               Mouse 13                                                                           4.3  0.82 0.67 0.75 0.54 1.1  <BKG <BKG --                               Mouse 14                                                                           4.1  0.80 0.70 0.76 0.54 0.33 <BKG <BKG --                               Mouse 15                                                                           3.1  0.73 0.65 1.1  0.50 0.44 <BKG <BKG --                               Mouse 16                                                                           0.64 0.28 0.62 0.93 <BKG <BKG <BKG <BKG --                               Mouse 17                                                                           0.54 0.21 0.67 0.96 <BKG <BKG <BKG <BKG --                               Mouse 18                                                                           0.59 0.30 0.48 0.61 <BKG <BKG <BKG <BKG --                               Mouse 19                                                                           3886.9                                                                             691.0                                                                              576.3                                                                              445.0                                                                              165.0                                                                              318.8                                                                              76.0 70.1 954.7                            Mouse 20                                                                           3115.6                                                                             464.8                                                                              309.5                                                                              242.7                                                                              134.8                                                                              230.0                                                                              170.4                                                                              81.9 1426.0                           Mouse 21                                                                           3592.8                                                                             675.0                                                                              478.3                                                                              439.0                                                                              157.8                                                                              335.2                                                                              198.0                                                                              166.5                                                                              1183.1                           Mouse 22                                                                           116.5                                                                              19.7 17.3 7.1  5.0  4.5  13.7 7.2  52.8                             Mouse 23                                                                           180.7                                                                              40.9 22.8 11.3 8.0  9.2  17.9 6.4  69.3                             Mouse 24                                                                           231.2                                                                              60.3 46.1 13.9 9.7  8.5  19.2 6.8  73.1                             Mouse 25                                                                           543.9                                                                              116.5                                                                              54.7 38.4 21.7 34.4 39.5 23.5 135.5                            Mouse 26                                                                           340.8                                                                              56.2 25.8 21.3 11.4 19.9 13.5 15.5 60.4                             Mouse 27                                                                           459.2                                                                              107.6                                                                              37.1 30.3 16.9 21.3 17.8 14.5 120.3                            Mouse 28                                                                           14.0 1.6  1.9  1.4  0.94 1.7  0.93 .68  5.0                              Mouse 29                                                                           9.9  1.3  1.4  8.2  0.61 0.87 0.75 .60  2.8                              Mouse 30                                                                           10.2 1.4  1.6  3.1  0.85 0.93 0.79 .63  3.4                              __________________________________________________________________________     Mice 1-3 and 19-21 = 500 μCi                                               adenosylocobalaminb-(4-aminobutyl)-amide-DTPA-.sup.111 In injected            intraperitoneal                                                               Mice 4-6 and 22-24 = 500 μCi DTPA.sup.111 In injected intraperitoneal      Mice 7-9 = 500 μCi                                                         adenosylcobalaminb-(4-amintobutyl)-amide-DTPA-.sup.111 In injected            subcutaneously                                                                Mice 10-12 = 500 μCi DTPA.sup.111 In injected subcutaneously               Mice 13-15 = approximately 30 μCi                                          methylcobalaminb-(4-aminobutyl)-amide-DTPA-.sup.111 In administered orall     Mice 16-18 = approximately 30 μCi DTPA.sup.111 In administered orally      Mice 25-27 = approximately 100 μCi                                         methylcobalaminb-(4-aminobutyl)-amide-DTPA-.sup.111 In tailvein injection     Mice 28-30 = approximately 100 μCi DTPA.sup.111 In tailvein injection 

Despite the difference in the amount of activity injected between IP andIV routes, the degree of uptake within the tumor was consistently secondbehind the kidneys. The tumors had two to four times greater activitythan the liver, spleen, and pancreas, with 4-12 times greater activitythan that of the heart, lungs, fat, and muscle. As expected, no activitywas seen to localize in the left flank of the control mice. Usual uptakein the liver and spleen was again seen. Gross pathology of the dissectedmasses demonstrated fat encapsulated tumors. Microscopically, by H & Estain, the tumors were solid masses of blue stained cells consistentwith a sarcoma. No areas of necrosis were noted.

Although DTPA-¹¹¹ In demonstrated uptake within the transplanted tumors,its concentration was 10-20 times less than that ofadenosylcobalamin-DTPA-¹¹¹ In.

D. Intravenous Administration: One milligram of either methyl oradenosylcobalamin-b-(4-aminobutyl)amide-DTPA was labeled with 5 mCi of^(99m) Tc as described above. Several mice were sacrificed via CO₂inhalation at varying time intervals after tailvein injection. The firsturine passed was collected and analyzed via TLC and AR.

E. Results

1. In Vivo Studies

(a) Biodistribution

The organ and tissue distribution of the methyl andadenosylcobalamin-DTPA analogs at 24 hours was similar despite the routeof administration (Table II). The kidneys were first, followed by theliver and spleen. The pancreas usually was next followed by the lungs,fat, heart, and muscle. The differences in activity between thepancreas, heart, lung, fat, and muscle was less significant after oral,subcutaneous and intravenous administration. However, the ratio ofuptake between the kidneys to liver, liver to spleen, and spleen topancreas was relatively constant. The route of administration (IV, IP,PO) did not have any obvious effect on the chelation of Tc-99m or In-111by these complexes.

The greatest amount of DTPA-¹¹¹ In uptake was in the kidneys. Thedistribution of DTPA was similar to the cobalamin analogs, especiallyafter intraperitoneal injection. Despite their similarities, DTPA-¹¹¹ Inhad 5-12 times less activity per organ or tissue sample when compared tothe methyl and adenosylcobalamin analogs.

(b) Gastrointestinal Absorption

Methylcobalarnin-b-(4-aminobutyl) amide-DTPA-In-111 was absorbed fromthe gastrointestinal tract after oral administration. The majority ofactivity was localized in the kidneys, liver, and spleen on delayedimaging. In the mice that were not "flushed" with oral andintraperitoneal doses of non-labeled methylcobalamin-b-(4-aminobutyl)amide-DTPA, no discernable activity was detected in the urine by gammawell counting. However, the mice that underwent the "modified Schillingstest" had detectable radioactivity within their urine at one hour.Imaging at 24 hours of these "flushed" mice demonstrated significantlyless activity throughout the body when compared to the "non-flushed"mice. Fecal radioactivity became detectable at 2 hours in both groupsreceiving the radioactive cobalamin analogs orally.

DTPA-¹¹¹ In was also absorbed from the gastrointestinal tract, but to alesser degree. No activity was detected in the heart, lungs, muscle, orfat tissue samples. Radioactivity was detected in urine and stool by twohours.

(c) Intravenous Administration

Micturition occurred at approximately 15 and 45 minutes afterintravenous and intraperitoneal injections, respectively. The firstpassed urine after intravenous or intraperitoneal administration wasalways radioactive. TLC and AR analysis of the collected urine showed noevidence of dissociation of the Tc-99m or In-111 from the cobalamin-DTPAcomplexes. Images at 5 minutes and 4 hours after tailvein injectiondemonstrated focal early uptake in the kidneys which became obscured bythe liver and spleen activity on the delayed images.

(d) Tumor Imaging

At 24 hours, there was a significant amount ofadenosylcobalamin-b-(4-aminobutyl) amide-DTPA-In-111 uptake within thetransplanted sarcoma both visually and by gamma well counting (TableII). Despite the difference in the amount of activity injected betweenIP and IV routes, the degree of uptake within the tumor was consistentlysecond behind the kidneys. The tumors had two to four times greateractivity than the liver, spleen, and pancreas, with 4-12 times greateractivity than that of the heart, lungs, fat, and muscle. As expected, noactivity was seen to localize in the left flank of the control mice.Usual uptake in the liver and spleen was again seen. Gross pathology ofthe dissected masses demonstrated fat encapsulated tumors.Microscopically, by H & E stain, the tumors were solid masses of bluestained cells consistent with a sarcoma. No areas of necrosis werenoted.

Although DTPA-¹¹¹ In demonstrated uptake within the transplanted tumors,its concentration was 10-20 times less than that ofadenosylcobalamin-DTPA-¹¹¹ In.

All publications, patents and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

What is claimed is:
 1. A compound of the formula: ##STR10## wherein themoiety ##STR11## is cobalamin, ##STR12## is the residue of amonocarboxylic acid of cobalamin, X is CN, OH, methyl or adenosyl, Y isa linking group and Det is a chelating group and a detectableradionuclide or a chelating group and a detectable paramagnetic metalion.
 2. The compound of claim 1 wherein the radionuclide is a metallicradioisotope.
 3. The compound of claim 2 wherein the metallicradioisotope is Tc^(99m), In¹¹¹ or Gd¹⁵³.
 4. The compound of claim 1wherein is the residue of the (b)-monocarboxylic acid.
 5. The compoundof claim 4 wherein Y is a divalent monomer, dimer or trimer ofN(H)(CH₂)2-6N(H).
 6. The compound of claim 5 wherein Y is --N(H)(CH₂)₄NH--.
 7. The compound of claim 1 wherein Det is EDTA, DTPA, DOTA, TETA,or DCTA.
 8. The compound of claim 3 wherein Det comprises DTPA.
 9. Amethod of evaluating kidney, liver, spleen or intestinal function in amammal comprising administering to said mammal a detectable amount of acompound of claim 2 in combination with a pharmaceutically acceptablevehicle, and detecting the presence of said compound in the kidney,liver, pancreas, spleen, or intestine of said mammal.
 10. The method ofclaim 9, wherein the administration is parenteral.
 11. The method ofclaim 10, wherein the administration is intravenous.
 12. The method ofclaim 11, wherein the administration is intraperitoneal.
 13. The methodof claim 9, wherein the administration is oral.